Measurement of components in blood, for example, antigens, antibodies, proteins, endocrine substances and so froth is clinically very important. In general, plasma or serum is used as a blood sample in many cases, and in such cases, whole blood is usually separated into serum or plasma as quickly as possible in order to avoid hemolysis. This is because, for example, in the field of immunological tests, if hemocytic components are present or hemolysis is caused in a sample, there may be caused disturbing phenomena such as influence of hemolysis on optical systems, inhibition of immunological reaction by internal components of blood cells and aggregation or adhesion of cytoplasmic membrane components of blood cells on an solid carrier used as a solid phase. Therefore, in ordinary clinical tests, it has been a common practice that extracted whole blood is first centrifuged to remove blood cells, and the obtained plasma or serum is used as a sample for measurement.
However, in order to remove blood cells, dedicated instruments such as centrifugal machine are required, and the operation is laborious. Therefore, it is desirable to use whole blood as it is as a measurement sample for physician in practice who do not have such installations and urgent tests with scarce temporal margin.
To satisfy the above requirements, there have been already proposed various methods of assaying whole blood itself without separating serum or plasma. As for immunoassay, a method utilizing latex coagulation as a homogeneous assay (method not requiring B/F separation) has been reported as a measurement method in which hemolysis of blood cells is intentionally and forcibly caused (Japanese Patent Laid-open Publication (Kokai) No. 10-48214). Secondly, as assay methods without causing hemolysis of blood cells, a homogeneous assay method using latex scattered light (clinical Chemistry, Vol. 43, 1764-1770 (1997)), a heterogeneous assay method (method requiring B/F separation) using a plastic cuvette as a solid phase (Japanese Patent Laid-open Publication No. 6-265554) and a method using polystyrene beads or magnetic particles as a solid phase (International Patent Unexamined Publication in Japanese (Kohyo) No. 2000-508075, WO96/04558) have been reported.
However, it cannot be said that convenient and highly sensitive assay methods using whole blood as a sample have already been established even by using these methods. First, even though some immunoassays using a homogeneous assay have been reported as convenient methods, an analyte is often a substance contained in blood in a trace amount in clinical tests and so forth, and therefore it is generally more strongly required to assay whole blood by using a heterogeneous assay that theoretically enables a highly sensitive assay. Secondly, with the background that solid carriers such as magnetic particles are widely used in a heterogeneous assay as a solid phase because of simplicity of B/F separation, micro particles such as magnetic particles are likely to be influenced by, in particular, blood cells, although solid carriers having a such size that the solid carriers should not aggregate causes no problem as in the cases of beads having a diameter of millimeter order and plastic plates. For example, when hemolysis occurs, inhibitory substances such as hemoglobin and cell nucleus-derived substances flowing out of the inside of blood cells into a reaction system may cause non-specific aggregation of solid carriers or reduce immune reaction, thereby seriously affecting the assay. Further, even when fresh unhemolyzed whole blood is used as a sample, if blood cells exist, solid carriers becomes likely to easily adhere on an inner wall of reaction vessel or a pipette tip due to blood cell membrane surface substances or the like, and thus harms such as inaccurate assay may be caused.
Moreover, instruments and cartridges for automatic assay are often used so as to quickly and conveniently conduct such assays of whole blood as described above. However, similar problems also occur in each step of such automatic assays. That is, since blood cell components in whole blood and solid carriers used for the assays are precipitated with time, it is essential to include a step of sufficiently stirring a sample containing whole blood prior to the assays so as to maintain blood cell components uniform or sufficiently stirring the sample, solid carrier, reagents etc. in a reaction step or assay step. In such a stirring step, a strong force is imposed on blood cells, and blood cells are disrupted, resulting in extremely easy hemolysis. Further, since suction and discharge of sample are performed in each step for successively transferring the sample to target reaction vessels following the steps, a strong force is imposed on blood cells and thus hemolysis easily occurs. In addition, non-specific adhesion and aggregation also easily occurs. Therefore, assay errors may be often caused.
In recent years, such instruments and cartridges for automatic assays as described above are also widely used in the field of point of care testing (POCT), which draws attentions as emergency test or test readily conducted by physicians and nurses. Accordingly, development of an assay method that can provide correct assay results even by an assay using such equipments and whole blood as it is has been required.